Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier

Villaseñor, Roberto; Ozmen, Laurence; Messaddeq, Nadia; Grüninger, Fiona; Loetscher, Hansruedi; Keller, Annika; Betsholtz, Christer; Freskgård, Per‐Ola; Collin, Ludovic

doi:10.1038/srep25658

Cited by 77 publications

(88 citation statements)

References 31 publications

(57 reference statements)

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“…Both uptake and transport of IgGs were altered in disease states, with the most significant increases in uptake and transport occurring with Aβ₁₋₄₂ incubation. The IgGs may be accumulating within the cells but are ultimately recycled back to the luminal compartment (Deane et al, 2005;Schlachetzki et al, 2002;Zhang & Pardridge, 2001) or are degraded within lysosomes (Villaseñor et al, 2016). Transport of IgGs was about twofold higher after incubation with Aβ₁₋₄₂, which is consistent with a study that showed a twofold increase in IgG transport after incubation with Aβ₁₋₄₂ in a primary murine BBB model (Wuest & Lee, 2014).…”

Section: Discussionsupporting

confidence: 85%

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid‐β and with neuroinflammatory cytokines

Mantle

Lee

2019

Biotech & Bioengineering

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Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood-brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulin G (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aβ₁₋₄₀ and Aβ₁₋₄₂) and neuroinflammation (tumor necrosis factor-α and interleukin-6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC-derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.

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Section: Discussionsupporting

confidence: 85%

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid‐β and with neuroinflammatory cytokines

Mantle

Lee

2019

Biotech & Bioengineering

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“…A recent study (Villaseñor et al . ) implicated Rab17‐dependent sorting microtubules in directing monovalent TfR antibody toward successful transcytosis; the bivalent TfR antibody‐TfR clusters were excluded from sorting microtubules by slower diffusion and were targeted for degradation. This hypothesis did not take into account the presence of various forms of the TfR on the surface of cells, some of which are naturally dimerized; nor was a range of affinities tested in a monovalent format.…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study, (Villaseñor et al . ) they showed a preferential sorting of a monovalent TfR antibody into sorting tubules which facilitated transcytosis across the BBB. However, a bivalent antibody that bound TfR in a pH‐sensitive fashion with lower affinity at acidic pH, typical of endosomal compartments, was found to escape degradation and release more efficiently on abluminal side of the BBB model in vitro (Sade et al .…”

mentioning

confidence: 94%

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood–brain barrier in vitro is dependent on its binding affinity

Haqqani

Thom²,

Burrell³

et al. 2018

Journal of Neurochemistry

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The blood–brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re‐directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter‐study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K d from 5 to174 nM) of the rat TfR‐binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics‐selected reaction monitoring and fluorescent imaging with markers of early‐ and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.Open Practices Open Science: This manuscript was awarded with the Open Materials Badge.For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

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“…Whereas the molecular mechanisms of endocytosis and recycling triggered by specific receptor-ligand interactions, notably transferrin binding to TfR, have been studied extensively in various cell types, 42,46 the intracellular sorting of antibodies that leads to their polarized transport and transcytosis across brain endothelium is less well understood. 47 Some specific pathways favoring transcytosis have started to emerge from this study; in particular, it appears that a subset of MVBs bearing markers of exocytosing MVBs may be an important pathway leading to abluminal exocytosis of the BBB-crossing FC5 antibody.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, A20.1Fc showed slightly higher P app values in vitro and measurable CSF levels in vivo compared to A20.1, suggesting that Fc fragment may contribute to low, but detectable non-specific penetration of antibodies into the brain described in several studies. 5,6,13 A recent study 42 using high-resolution quantitative microscopy showed that lysosomal degradation of low levels of IgGs internalized into BEC from systemic circulations may be further limiting the antibody access to the brain.…”

Section: Discussionmentioning

confidence: 99%

Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

Haqqani

Delaney

Brunette

et al. 2017

J Cereb Blood Flow Metab

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Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=33302487-9987-42d0-bb7f-3b59445d5f72 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=33302487-9987-42d0-bb7f-3b59445d5f72 AbstractCurrent methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody 'tracking' in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody 'redistribution' from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

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Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier

Cited by 77 publications

References 31 publications

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid‐β and with neuroinflammatory cytokines

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid‐β and with neuroinflammatory cytokines

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood–brain barrier in vitro is dependent on its binding affinity

Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

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